Methods of use related to xerostomia

ABSTRACT

The present invention relates to new uses of thermally stable, crystalline S-2-(3-aminopropylamino)ethyl dihydrogen phosphorothioate, (amifostine) and other aminothiol compounds to treat and reverse toxicities caused by radiation treatment. In particular, the invention provides a method for treating or preventing xerostomia associated with the administration of radiation treatment of head and neck cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/137,686, filed on May 3, 2002, which is a division of applicationSer. No. 09/429,290, filed on Oct. 28, 1999, now U.S. Pat. No.6,586,476, which is a division of application Ser. No. 08/987,550, filedon Dec. 9, 1997, now U.S. Pat. No. 5,994,409. The above-referencedpatent applications and patents are herein incorporated by reference intheir entirety.

1. FIELD OF THE INVENTION

The present invention relates to new uses for thermally stable, sterile,crystalline S-2-(3-aminopropylamino)ethyl dihydrogen phosphorothioate,also known as amifostine, its salts, hydrates, esters, metabolites,functional derivatives, functional analogues, and related aminothiolcompounds, to treat or prevent xerostomia caused by radiation therapy ofhead and neck cancer.

2. BACKGROUND OF THE INVENTION

2.1. Aminothiol Compounds

The compound S-2-(3-aminopropylamino)ethyl dihydrogen phosphorothioate(which is also known as amifostine, ethiofos, Ethyol®, NSC 296961, andWR-2721 and which will hereinafter be referred to as “amifostine”) andother aminothiol compounds are disclosed in U.S. Pat. No. 3,892,824 toPiper et al. These compounds were originally developed as antiradiationagents (radio-protectants), in particular to be used prior to exposureto x-ray or nuclear radiation, to protect against the harmful effects ofsuch exposure which may be encountered during military conflicts.

In addition to its utility as a military antiradiation agent, amifostinehas demonstrated excellent utility as a non-military radioprotectant andchemoprotectant, i.e., as a protectant administered prior to therapy toreduce the undesirable adverse effects which arise during the use ofchemotherapy and radiation therapy in the treatment of cancer. Nygaardet al., eds., 1983, Radioprotectors and Anticarcinogens, Academic Press,Inc., New York, pp. 73-85; Grdina et al., 1985, “Radioprotector WR-1065Reduces Radiation-Induced Mutations of the HGPRT Locus in V79 Cells,”Carcinogenesis (London) 6:929-931. In addition, these compounds havebeen reported to afford protection against the adverse effects ofchemotherapeutic agents, for example, alkylating agents such ascisplatin and carboplatin, when administered before or concurrently withthe chemotherapeutic agent. Jordan et al., 1982, “Modulation ofcis-platinum renal toxicity by the Radioprotective agent WR-2721”, Exp.Mol. Pathol. 36:297; Doz et al., 1991, “Experimental Basis forIncreasing the Therapeutic Index of Carboplatin in Brain Tumor Therapyby Pretreatment With WR-Compounds”, Cancer Chemother. Pharmacol. 28:308.Similarly, it has been reported that amifostine has been usedexperimentally prior to therapy to protect HIV-infected patients (AIDS)from the harmful side effects of 3′-azido-3′-deoxythymidine (AZT)therapy. International Published Application WO 90/14007, published Nov.29, 1990. Amifostine and its derivatives have been shown to exert thesereported protective effects without affecting the beneficial propertiesof the administered therapeutic agents. This is, in the case ofchemotherapy, believed to be due to the selective uptake of theprotective thiol and other metabolites into normal tissue. Yuhas, 1980,“Active versus Passive Absorption Kinetics as the basis for SelectiveProtection of Normal Tissues by WR-2721”, Cancer Res. 40:1519-1524;Yuhas, 1979, “Differential Protection of Normal and Malignant TissuesAgainst the Cytotoxic Effects of Mechlorethamine” Cancer Treat. Rep.63:971-976.

Amifostine and related aminothiol compounds have also been shown tostimulate bone marrow growth. See U.S. patent application Ser. No.08/390,713; International Published Application WO 96/25045 publishedAug. 22, 1996; List et al., “Amifostine Stimulated Formation ofMultipotent Progenitor and Generated Macroscopic Colonies in Normal andMyelodysplastic Bone Marrow,” Proc. Am. Soc. Clin. Oncol. 15:449 [1403][Abstract]. Currently, amifostine is in Phase II clinical trials as abone marrow stimulant in patients suffering from myelodysplasticsyndrome. List et al., 1996, “Amifostine Promotes MultilineageHematopoiesis in Patients with Myelodysplastic Syndrome (MDS): Resultsof a Phase I/II Clinical Trial,” Am. J. Hem. 1 (Abstract); List et al.,1996, “Amifostine Promotes in vitro and in vivo Hematopoiesis inMyelodysplastic Syndromes,” Chem. Found Sympos. (Abstract); List et al.,1996, “Amifostine Promotes Multilineage Hematopoiesis in Patients withMyelodysplastic Syndrome (MDS): Results of a Phase I/II Clinical Trial,”Abstract, 8th Annual Meeting, American Society of Hematology, Orlando,Fla. Pre-exposure with aminothiol compounds is capable of causing thebone marrow function to more rapidly recover following chemotherapy.List et al., 1996, “Amifostine Protects Primitive HematopoieticProgenitors Against Chemotherapy Cytotoxicity,” Semin. Oncol. 23 (4),Supp. 8:58-63.

Presently, amifostine is indicated to reduce the cumulative renaltoxicity associated with repeated administration of cisplatin inpatients with advanced ovarian or non-small cell lung cancer.Physicians' Desk Reference, 51 st ed. 1997, p. 485-486. The recommendedstarting dosage for adults in an FDA-approved indication is 910 mg/m²administered once daily as a 15-minute intravenous (i.v.) infusion,starting 30 minutes prior to chemotherapy. However, clinical trials haveused doses as low as 100 mg.

U.S. Pat. Nos. 5,567,686 and 5,488,042, both to Grdina, allege that theadministration of an aminothiol compound before irradiation of a mammalaffords protection against genotoxic mutagenesis. Although the U.S. Pat.No. 5,488,042 discloses administering an aminothiol up to about threehours after irradiation, both patents focus solely on prevention ofmutations, rather than the treatment or reversal of radiation- orchemotherapy-induced damage. Further, the Grdina patents are silent asto the use of any aminothiol compound in humans to treat chemotherapy-or radiation-induced disorders and toxicities including neuro-, nephro-,hematological or mucosal disorders.

Nagy et al., 1986, “Protection Against cis-DiamminedichloroplatinumCytotoxicity and Mutagenicity in V79 Cells by2-[(Aminopropyl)amino]ethanthiol,” Cancer Research 46:1132-1135,disclose that the free thiol metabolite of amifostine, also known asWR-1065, protects against cytotoxicity in V79-B310H Chinese hamstercells when administered before, during and immediately after treatmentof the cells with cis-diamminedichloroplatinum (cis-DDP, “cisplatin”).Although some protection against cell death was observed under allconditions, Nagy et al. report that maximum protection was obtained whenWR-1065 was present in the cell growth medium for 30 minutes prior toexposure of cells to cisplatin. In addition, Nagy et al. state thatlittle if any difference in the magnitude of protection against cellkilling was seen whether the WR-1065 was present either during orimmediately following cisplatin exposure.

Treskes et al., 1992, “Effects of the Modulating Agent WR-2721 and itsMain Metabolites on the Formation and Stability of Cisplatin-DNA Adductsin Vitro in Comparison to the Effects of Thiosulphate andDiethyldithiocarbonate,” Biochemical Pharmacology 43(5): 1013-1019investigated the ability of amifostine and its main metabolites, WR-1065and WR-33278, to prevent formation of adducts of cisplatin with the DNAof salmon sperm. They found that amifostine, WR-1065 and WR-33278 causeda decrease in the platination of salmon sperm in vitro when thecompounds were present concomitantly with cisplatin. It was alsoobserved that part of the already formed cisplatin-DNA adducts isdisrupted during post-incubation with subject compounds, but thisdecrease in adduct levels was small compared to those obtained duringco-incubations. Treskes et al. speculated that conformational changes inDNA induced by the WR-1065 metabolite of amifostine observed by otherresearchers might provide a rational for applying amifostine aftercisplatin administration.

However, Treskes et al. later reported in 1993, “WR2721 as a Modulatorof Cisplatin- and Carboplatin-induced Side Effects in Comparison withOther Chemoprotective Agents: A Molecular Approach,” Cancer Chemother.Pharmacol. 33:93-106, that neither WR-1065 nor WR-2721 (amifostine)could protect cells from the cytostatic effect of cisplatin when thecompounds were incubated with cells one hour after cisplatin exposure.Further, it was found that amifostine given 30 minutes after cisplatindid not protect mice at all from nephrotoxicity. Treskes et al.concluded that the selective protection of multiple non-tumor tissue byWR-1065 from cisplatin-induced toxicity is explained by a strongprevention, not reversal, of cisplatin-induced cellular damage. Treskeset al. further observed that these findings were in agreement with thehypothesis that the prevention of damage is the main mechanism ofprotection and that reversal of platinum-induced damage is not animportant mechanism of protection.

Nephrotoxicity, produced by drugs such as cisplatin, has importantconsequences for the patient, with potential permanent loss of 50% ormore of normal renal function (Kemp, et al. J. Clin. Oncology,14:2101-2112, July, 1996). This can produce serious disability,requiring the need for dialysis in severe cases, and early mortality. Italso has important consequences for the ability of the patient to besafely treated with other forms of life-sustaining chemotherapy andother medications such as antibiotics that are themselves renally toxicor require adequate renal function for elimination from the body.

Neurotoxicity may significantly decrease a patient's quality of lifebecause of loss or distortion of sensation in the fingers, toes, handsand feet, as well as loss of fine muscle movements, resulting in theinability to perform routine functions such as buttoning of clothes. Inmore severe cases, patients suffer loss of sufficient motor function sothat they require walkers or wheelchairs.

2.2. Cisplatin- and Paclitaxel-Induced Toxicities

Cisplatin continues to be an agent of choice for the treatment ofadvanced ovarian cancer, testicular cancer, bladder cancer and head,neck and lung cancers. McGuire et al., 1996, “Cyclophosphamide andcisplatin compared with Paclitaxel and cisplatin in patients with stageIII and stage IV ovarian cancer,” N. Engl. J. Med. 334:1-6. However, thecytotoxic effects of cisplatin on normal tissue, including the kidneys,can result in long-term debilitating effects which may limit the abilityto deliver therapeutic doses against the cancer. Despite aggressivehydration and administration of mannitol, cisplatin-inducednephrotoxicity remains a significant cause of morbidity and mortality.Finley et al., 1985, “Cisplatin nephrotoxicity: A Summary ofPreventative Interventions,” Drug Intell. Clin. Pharm. 19:362-367; Kempet al., 1996, “Amifostine Pretreatment of Protection AgainstCyclophosamide-induced and cisplatin-induced toxicities: results of arandomized control trial in patients with advanced ovarian cancer,” J.Clin. Oncol. 14:2101-2112; Stewart et al., “Association of CisplatinNephrotoxicity with patient Characteristics and Cisplatin AdministrationMethods,” Cancer Chemother. Pharmacol. 40:293-308.

Even when protective agents are administered prior to or duringcisplatin treatment, toxicities may still be observed. The cumulativecisplatin nephro-toxicity that occurs can be treatment-limiting,precluding further administration of cisplatin and the ability toadminister effective doses of renally-excreted second-line chemotherapyKemp et al., 1996, J. Clin. Oncol. 14:2101-2112. In addition, drugtherapy for other conditions can be affected, either because the agentsare renally excreted, or because of their intrinsic potential to worsenrenal function. Nephrotoxicity is also associated with other platinumcoordination complexes such as carboplatin which are also used to treatcancers.

The nephrotoxicity associated with cisplatin is cumulative, i.e.,incremental damage occurs with repeated courses of therapy. Daugaard etal., 1989, “Cisplatin nephrotoxicity,” Cancer Chemother. Pharmacol.21:1; du Bois, et al., 1994, “Cisplatin and carboplatin induced acute,cumulative and chronic nephrotoxicity,” Proc. Annu. Meet. Am. Assoc.Cancer Res. 35: A1475. Loss of renal function occurring secondary tocisplatin is typically permanent. Macleod et al., 1988, “The effect ofcisplatin on renal function in patients with testicular tumors,” Clin.Radiol. 39: 190-192; Aass et al., 1990, “Renal function related todifferent treatment modalities for malignant germ cell tumors,” Br. J.Cancer 62:842-846; Meijer, et al., 1983, “Influence of combinationchemotherapy with cisdiammine chloroplatinum on renal function: Longterm effects,” Oncology 40:170-173. Risk factors for development of thistoxic effect include age, a history of renal irradiation, dehydration,and alcoholism. Anand et al., 1993, “Newer insights into cisplatinnephrotoxicity,” Ann. Pharmacother. 27:1519-1525.

Nephrotoxicity associated with administration of cisplatin and otherplatinum coordination complexes is generally observed during the secondweek after a dose and is manifested by elevations in BUN and/or serumcreatinine and/or a decrease in creatinine clearance or serum magnesium.Renal toxicity becomes more prolonged and severe with repeated coursesof the drug. Nephrotoxicity may be accentuated in patients withpre-existing risk factors such as diabetes or hypertension as well asindividuals who are receiving other nephrotoxins such as aminoglycosideantibiotics or antifungals such as amphoterecin. Options available toreduce cumulative cisplatin renal toxicity are limited, and generallyinvolve reducing its dosage or frequency of administration, both ofwhich risk a potential reduction in antitumor effectiveness. Kemp etal., 1996, J. Clin. Oncol. 14:2101-2112. Pretreatment or simultaneousadministration of amifostine is also an option, but these treatments arenot always effective.

In addition to nephrotoxicity, many other toxicities are associated withthe administration of platinum coordination compounds. For example,ototoxicity has been observed in up to 31% of patients treated with asingle dose of cisplatin (50 mg/m²) and is manifested by tinnitus and/orhearing loss in the high frequency range (4000-8000 Hz). Decreasedability to hear normal conversational tones may occur occasionally.Deafness after the initial dose of cisplatin has been reported rarely.Hearing loss can be unilateral or bilateral and tends to become morefrequent and severe with repeated doses. In addition, myelosuppressionoccurs in 25-30% of patients treated with cisplatin. The nadirs incirculating platelets and leukocytes occur between days 18 to 23 withmost patients recovering by day 39. Leukopenia and thrombocytopenia areobserved at higher doses (>50 mg/m²).

Neurotoxicity, usually characterized by peripheral neuropathies, is alsoassociated with administration of platinum coordination complexes. Theneuropathies, in the form of loss or distortion of sensation, or loss offine motor function, usually occur after prolonged therapy (4-7 months);however, neurologic symptoms have been reported to occur after a singledose. Although symptoms and signs of cisplatin-induced neuropathyusually develop during treatment, symptoms of neuropathy may begin 3 to8 weeks after the last dose of the platinum coordination complex.Generally, in the event of cisplatin-induced neuropathic symptoms,cisplatin is discontinued until the symptoms subside or disappear. Theneuropathy, however, may progress even after treatment is stopped. Somepreliminary evidence suggests that neuropathy may be irreversible insome patients. Lhermitte's sign, dorsal column myelopathy, autonomicneuropathy, loss of taste and seizures have also been reported. Musclecramps, defined as localized, painful, involuntary skeletal musclecontractions of sudden onset and short duration, or loss of sufficientmotor function so that a patient requires a walker or wheel chair formovement, have been reported and are usually associated in patientsreceiving a relatively high cumulative dose of cisplatin and exhibitingadvanced symptomatic stages of peripheral neuropathy. Amifostine hasdemonstrated the ability to reduce the incidence of neuropathies whenadministered prior to cisplatin. In a prospective evaluation of patientstreated with cisplatin regimens±amifostine at the Hospital of theUniversity of Pennsylvania Cancer Center, patients pretreated withamifostine had a significantly lower incidence of cisplatin neuropathiesand the onset of neuropathies occurred at a significantly highercumulative dose of cisplatin [Mollman J E, Glover D J, Hogan W M, FurmanR E: Cisplatin neuropathy: Risk factors, prognosis and protection byWR-2721. Cancer 61:2192-2195, 1988]. The ability of amifostinepretreatment to significantly reduce the occurrence and severity ofcisplatin neuropathy was confirmed in a randomized controlled trial ofcisplatin and cyclophosphamide±amifostine in women with advanced ovariancancer [Kemp G, Rose P, Lurain J et al.: Amifostine pretreatment forprotection against cyclophosphamide and cisplatin induced toxicities:Results of a randomized control trial in patients with advanced ovariancancer. J. Clin. Oncol. 14:2101-2112, 1996].

Paclitaxel is indicated, after failure of first-line or subsequentchemotherapy, for the treatment of metastatic carcinoma of the ovary.Paclitaxel is also indicated for the treatment of breast cancer afterfailure of combination therapy for metastatic disease or relapse withinsix months of adjuvant therapy. Paclitaxel is known to have thefollowing adverse effects: neutropenia, leukopenia, peripheralneuropathy, and arthralgia/myalgias and other neurologicalmanifestations. The results of a clinical trial of amifostine andescalating doses of paclitaxel, indicated that pretreatment withamifostine allowed both higher single doses and cumulative doses ofpaclitaxel to be administered without the occurrence of dose limitingneuropathies and arthralgias/myalgias [DiPaolo R et al.: Amifostine anddose intense paclitaxel in patients with advanced malignancies. CancerTherapeutics, Proc. Am. Soc. Clin. Oncology Vol. 16 (Abstract 826) 235a(1997)]. These may be accentuated when the drug is combined with otherneurotoxic agents such as cisplatin. Many patients receiving paclitaxelalso experience hypotension, asymptomatic bradycardia, and occasionalepisodes of silent ventricular tachycardia.

Hence, there is a need for a chemical agent for treating symptoms ofneurotoxicity and nephrotoxicity which result from the administration ofcertain therapeutic agents, particularly, chemotherapeutics, radiationtherapy, or disease states such as diabetes.

3. SUMMARY OF THE INVENTION

The present invention relates, in part, to methods of treatingxerostomia in a human, said xerostomia associate with radiation therapyfor head and neck cancer, which comprises administering to said human atherapeutically effective amount of a dosage form comprising one or moreaminothiol compounds, or pharmaceutically acceptable salts thereof, tothe human after the occurrence of one more of the toxicities.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing serum creatinine levels (mg/dL) of a61-year-old patient suffering from renal failure induced by cisplatingiven in combination with paclitaxel. The graph shows a starting serumcreatinine level of about 1.0 mg/dL before administration of cisplatin,a maximum level of about 5.5 mg/dL after administration of cisplatin,and a drop in serum creatinine level to about 2.8 mg/dL followingtreatment with amifostine.

FIG. 2 depicts the molecular and crystal structure of vacuum driedamifostine.

FIG. 3 depicts the X-ray diffraction of crystalline amifostine drugproduct formulated from 10% ethanol in water.

FIG. 4 depicts the X-ray diffraction of amifostine drug substance.

5. DETAILED DESCRIPTION OF THE INVENTION

There are presently a number of chemotherapeutic agents that can be usedagainst various cancers, including solid tumors and leukemias.Unfortunately, these chemotherapeutics frequently cause adverse orundesirable side effects which limit the clinician's ability to use thedrug effectively. Most significantly, chemotherapeutics can cause tissuedamage, organ damage and the like, which is not only painful to thepatient but can also be irreversible or lethal depending upon thepatient's tolerance and condition.

The inventors have quite surprisingly and unexpectedly found that theadministration of amifostine, and related aminothiol compounds disclosedherein, can be used to reverse or treat the toxicities associated withthe administration of various chemotherapeutics, particularly platinumcoordination complexes such as cisplatin and paclitaxel. Morespecifically, upon clinical evidence of neuro- or nephrotoxicitycommonly associated with the administration of cisplatin or paclitaxel,amifostine or related compounds can be used to rescue the patientthereby improving the overall treatment/therapy.

Based in part on this discovery, the present invention encompasses amethod of treating neuro- or nephro-disorders in a human which comprisesadministering a therapeutically effective amount of amifostine, or apharmaceutically acceptable salt, ester, analogue, metabolite,derivative or pro-drug thereof. The neuro- and nephro-toxicities whichare treatable according to the methods of the present invention canarise from a variety of insults including, but not limited to cancerchemotherapy, radiation therapy, AIDS, chemotherapy, anti-fungaltherapy, anti-bacterial therapy, and I.V. Contrast Agents. Theseaminothiol compounds can also be used to treat neuro- andnephro-disorders that are induced by aging and metabolic disorders,including, but not limited to diabetes. The methods of the presentinvention are also suitable for the treatment of neuro- andhephro-disorders induced by an unknown etiology. The methods of theinvention are effective for the treatment of patients with and withoutcancer, as well as cancer patients undergoing or who have undergonechemotherapy. It should be recognized that the present invention alsoencompasses a method of treating various cancers by the combined use ofa chemotherapeutic agent and one or more of the aminothiol rescueagents, such as amifostine, disclosed herein. Indeed, the use of theserescue agents allows the continued use of the chemotherapeutic agentwhich would have otherwise been discontinued or postponed due totoxicity.

In another embodiment, the present invention provides a method fortreating toxicities associated with administration of a therapeuticagent wherein amifostine, or a related compound is administeredsubsequent to administration of the therapeutic agent. Theadministration of amifostine, and the compounds disclosed herein, afterthe occurrence of toxicities associated with administration of thetherapeutic agent ameliorates and reverses the signs and symptoms ofthese toxicities. Thus, the present invention encompasses methods fortreating toxicities associated with chemotherapy by administeringamifostine, or salts, metabolites, functional derivatives functionalanalogues, esters and pro-drugs thereof after therapeutic treatment.

The present invention further provides a method for treating toxicitiesassociated with the administration of a chemotherapeutic agent whichcomprises administering a therapeutically effective amount of amifostineor a metabolite, functional derivative or analogue thereof, orpharmaceutically acceptable salts thereof after the occurrence of one ormore of said toxicities.

In particular, the present invention provides a method for treatingneurotoxicity and nephrotoxicity associated with the administration ofcisplatin or paclitaxel agent which comprises administering atherapeutically effective amount of amifostine or a salt, metabolite,ester, functional derivative, functional analogue and pro-drugs thereof,subsequent to the administration of the chemotherapeutic agent. In apreferred embodiment, the compound is administered after the disorderhas appeared and been established, typically one or more days afteradministration of the chemotherapeutic agent.

The invention also encompasses the use of the aminothiols for thespecific treatment of peripheral neuropathy, central neuropathy,autonomic neuropathy, muscle weakness, and myalgaia.

5.1. Aminothiols and Related Compounds that are Useful within theInvention

As mentioned above, the compounds that can be used within the presentinvention include amifostine (WR-2721), as well as salts, hydrates,active metabolites, pro-drugs, and functional derivatives or analogues.More specifically, the invention includes all pro-drugs and metabolitesof amifostine and pro-drugs of the active metabolites. Thus, compoundsknown to the skilled artisan to be suitable for administration to humansand known to be metabolites or otherwise converted into active thiolsincluding metabolites such as WR-1065 and WR-33278 (disulfide) and theorally bioavailable WR-151327 and its active thiols, includingmetabolites such as WR-151326 and its corresponding disulfide, areencompassed within the present invention.

Similarly, described herein are aminothiols that exhibit activitysimilar to that of amifostine or its metabolites. Preferably, thesecompounds are structurally related to amifostine. Alternatively, thesecompounds are pro-drugs that are metabolized in vivo to a biologicallyactive agent. These compounds are also encompassed by the presentinvention. Specific examples are illustrated herein.

Aminothiol compounds which can be used in the present invention arerepresented by the following formula (I):R₁NH(CH₂)_(n)NH(CH₂)_(m)SR₂  (I)wherein R₁ is hydrogen, C₅-C₇ aryl, C₂-C₇ acyl, or C₁-C₇ alkyl; R₂ ishydrogen, PO₃H₂ or R₃, wherein R₃ is R₁NH(CH₂)_(n)NH(CH₂)_(m)S—; n and mare each an integer from 1 to 10; and preferably an integer from 2 to 6.

The methods of the present invention also encompasses the use ofpharmaceutically acceptable salts and hydrates of the compounds offormula (I) above.

Preferred compounds useful in the methods of the invention are theS-ω(ω-amino-alkylamino)alkyl dihydrogen phosphorothioate analoguesrepresented by the formula:R—NH—(C_(n)H_(2n))—NH—(C_(m)H_(2m))—S—PO₃H₂wherein R is hydrogen or an alkyl group containing 1 to 7 carbon atomsand m and n independently have a value of from 1 to 10, preferably 2 to6.

The chemical structure of amifostine (WR-2721) can be depicted asfollows:H₂N—(CH₂)₃—NH—(CH₂)₂—S—PO₃H₂.

One preferred metabolite of amifostine is a dephosphorylated free thiolform known as WR-1065 (chemical nomenclature: S-2-(3-aminopropylamino)ethanethiol), which can be depicted as follows:H₂N—(CH₂)₃—NH(CH₂)₂—SH.

Another preferred metabolite of amifostine is its disulfide, known asWR-33278 (chemical nomenclature:[2-[(aminopropyl)amino]ethanthiol]-N,N′-dithioidi-2,1-ethanediyl)bis-1,3-propanediamine),which can be depicted as follows:H₂N—(CH₂)₃—NH—(CH₂)₂—S—S—(CH₂)₂—NH—(CH₂)₃—NH₂.

A preferred analogue of amifostine is the compound designated asWR-15327 (chemical nomenclature:1-propanethiol-3-[[3-(methylamino)propyl]amino]-dihydrogenphosphothiorate), which can be depicted as follows:CH₃NH(CH₂)₃NH(CH₂)₃SPO₃H₂.

Another preferred analogue of amifostine is the compound designatedWR-151326, a dephosphorylated free thiol form of WR-151327 having thechemical structure:CH₃NH(CH₂)₃NH(CH₂)₃SH.

Other specific compounds suitable for use in the present inventioninclude, but are not limited to:

-   S-1-(aminoethyl)phosphorothioic acid (WR-638);-   S-[2-(3-methylaminopropyl)aminoethyl]phosphorothioate acid    (WR-3689);-   S-2-(4-aminobutylamino)ethyl phosphorothioic acid (WR-2822);-   3-[(2-mercaptoethyl)amino]propionamide p-toluene-sulfonate    (WR-2529);-   S-1-(2-hydroxy-3-amino)propyl phosphorothioic acid (WR-77913);-   2-[3-(methylamino)propylamino]ethanethiol (WR-255591);-   S-2-(5-aminopentylamino)ethyl phosphorothioic acid (WR-2823);-   1-[3-(3-aminopropyl)thiazolidin-2-Y1]-D-gluco-1,2,3,4,5    pentane-pentol dihydrochloride (WR-255709).

Additional aminothiols suitable for use in the present inventioninclude, but are not limited to, S-2-(3-ethylaminopropylamino)ethyldihydrogen phosphorothioate, S-2-(3-aminopropylamino)-2-methylpropyldihydrogen phosphorothioate, S-2-(2-aminoethylamino)-2-ethyl dihydrogenphosphorothioate, S-2-(4-aminobutylamino)-2-ethyl dihydrogenphosphorothioate, S-2-(5-aminopentylamino)-2-ethyl dihydrogenphosphorothioate, S-2-(6-aminohexylamino)-2-ethyl dihydrogenphosphorothioate, S-2-(2-methylaminoethylamino)-2-ethyl dihydrogenphosphorothioate, S-2-(3-methylaminopropylamino)-2-ethyl dihydrogenphosphorothioate, and S-3-(3-methylamino-propylamino)-3-propyldihydrogen phosphorothioate (WR-151327) and pharmaceutically acceptablesalts thereof. Preferably, the aminothiol is amifostine, WR-1065,WR-33278, WR-151327 or WR-151326; most preferably it is amifostine.

Amifostine, and many of its salts, analogues and derivatives thereofsuitable for use in the methods of the invention are commerciallyavailable, or can easily be prepared using standard techniques. Theaminothiol compounds useful in the methods of the invention may beprepared by methods known in the art (see, e.g., Cortese, 1943, OrganicSynthesis pp. 91-93, Coll. Vol. II, Blatt, Ed., John Wiley & Sons, Inc.,New York, N.Y.; Akerfeldt, 1960, Acta Chem. Scand. 14:1980; Piper etal., 1966, Chem. Ind. (London):2010). Certain aminothiol compounds, aswell as methods of synthesizing such compounds, are described in detailin U.S. Pat. No. 3,892,824 to Piper et al., U.S. Pat. Nos. 5,424,472 and5,591,731, both to Kennedy et al., and WO 96/25045, each of which isincorporated herein by reference in its entirety.

The aminothiol compounds useful in the methods of the invention may bein the form of free acids, free bases, or pharmaceutically acceptableaddition salts thereof. Such salts can be readily prepared by treatingan aminothiol compound with an appropriate acid and/or base. Such acidsinclude, by way of example and not limitation, inorganic acids such ashydrohalic acids (hydrochloric, hydrobromic, hydrofluoric, etc.),sulfuric acid, nitric acid, phosphoric acid, etc. and organic acids suchas acetic acid, propanoic acid, 2-hydroxyacetic acid, 2-hydroxypropanoicacid, 2-oxopropanoic acid, propandioic acid, butandioic acid, etc.Conversely, the salt can be converted into the free base form bytreatment with alkali.

The aminothiol compounds useful in the methods of the invention, as wellas the pharmaceutically acceptable addition salts thereof, may be in ahydrated, solvated or anhydrous form. Methods of preparing such formswill be apparent to those of skill in the art of organic chemistry.

5.2. Definitions

As used herein the term “aminothiol” means a compound represented byformula (I) set forth in Section 5.1 above, or any other compounddisclosed therein.

The term “rescue agent” as used herein is intended to mean a compoundcapable of ameliorating, treating, reversing, reducing or arresting thesigns and symptoms and pathology associated with the administration ofchemotherapeutic agents radiation and pathology of associated diseases.

As used herein the term “disorder” means an illness, a sickness or adisease manifested by an interruption, cessation, derangement orabnormality of body functions, systems or organs.

As used herein, the term “toxicity” means a disorder characterized by arecognized etiologic agent or agents, an identifiable group of signs andsymptoms, including adverse effects, unwanted effects, undesiredeffects, or abnormal signs or symptoms or consistent anatomicalalterations.

The preferred subjects of the present invention are mammals, includinghumans. The subjects include cancer patients that are undergoing or haveundergone chemotherapy, radiation treatment or both; AIDS patients anddiabetics.

The term “treating” as used herein is intended to mean the administeringto a subject the rescue agent of the present invention, preferablyamifostine or a functional analogue or derivative thereof, for purposeswhich may include amelioration of symptoms of, or reversal of toxicitiesassociated with chemotherapy.

As used herein the term “reversing” means that the progress of thedisease, disorder or toxicity is inhibited and its symptoms are reversedor improved.

5.3. Toxicities/Disorders to be Treated

The methods of the present invention comprise administration of apharmaceutical composition which contains an effective amount of anaminothiol of the invention in an acceptable carrier to a subject duringor preferably after the subject has received therapy. The aminothiol ispreferably amifostine, alone or in combination with one or more otherdrugs useful in the treatment of toxicities associated with therapy.Also included within the scope of the invention is the administration ofcompositions comprising a mixture of two or more of the aminothiolcompounds of the present invention described above.

The methods of the present invention are suitable for treatingtoxicities associated with a wide variety of therapeutic agents. Inaddition, the methods of the invention are suitable for treatment of avariety of neuro- and nephro-disorders resulting from a variety ofinsults.

5.3.1 Chemically- and Radiation-Induced Toxicities

In one embodiment, the methods of the invention are used to treattoxicities associated with administration of chemotherapeutic agentswhich include, but are not limited to, cisplatin, carboplatin,paclitaxel, vinblastine, vincristine and methotrexate. In anotherembodiment, the methods of the invention are used to treat toxicitiesassociated with radiation therapy (x-ray, nuclear and particularly gammaradiation).

The methods of the invention may be used to treat toxicities associatedwith the administration of the following chemotherapeutic agents:taxanes such as paclitaxel and docetaxel; alkylating agents, whichinclude: nitrogen mustards such as mechlorethamine, cyclophosphamide,ifosamide, melphalan (phenalphenine mustard) and chlorambucil;ethylenimines and methylmelamines such as altretamine, diaziquone (AZQ)and thiotepa; alkyl sulfonates such as busulfan; nitrosoureas such ascarmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU) andstreptozocin (streptozotocin); and triazenes such as dacarbazine (DTIC;dimethyltriazenoimidazolecarboxamide); antimetabolites, which includefolic acid analogs such as methotrexate, trimetrexate and otherdihydrofolates; pyrimidine analogs such as fluorouracil (5-fluorouracil;5-FU), floxuridine (fluorodeoxyuridine; FUdR) and cytarabine (cytosinearabinoside); purine analogs and related inhibitors such asmercaptopurine (6-mercaptopurine; 6-MP), thioguanine(6-thioguanine; TG)and pentostatin (2′-deoxycoformycin); natural products, which includevinca alkaloids such as vinblastine, vincristine, navelbine andvincristine; epipodophylotoxins such as etoposide and teniposide;antibiotics such as dactinomycin (actinomycin D), daunorubincin(daunomycin; rubidomycin); doxorubicin, bleomycin, plicamycin(mithramycin) and mitomycin (mitomycin C); enzymes such asL-asparaginase; and biological response modifiers such asinterferon-alfa and other interferons; platinum coordination complexessuch as cisplatin (cis-DDP) and carboplatin; anthracenediones such asmitoxantrone; substituted ureas such as hydroxyurea; methylhydrazidederivatives such as procarbazine (N-methylhydrazine, MIH), andadrenocortical suppressants such as mitotane (o,p′-DDD) andaminoglutethimide; hormones and antagonists which includeadrenocorticosteroids such as prednisone; progestins such ashydroxyprogesterone caproate, medroxy progesterone acetate and megestrolacetate; estrogens such as diethylstilbestrol and ethinyl estradiol;antiestrogens such as tamoxifen; androgens such as testosteronepropionate and fluoxymetsterone; antiandrogens such as flutamide; andgonadotropin-releasing hormone analogs such as leuprolide; camptothecinssuch as irinotecan, topotecan; gemciatdins such as gemcitabine;estramustine phosphate, VM-26 (vumon) and all-trans retinoic acid(ATRA). These agents are normally used in the treatment of head andneck, ovarian, breast, colon, lung, prostate, testicular and cervicalcancers, as well as certain lymphomas, leukemias, and cancers of theCNS.

The toxicities associated with the administration of these agents orradiation therapy include, but are not limited to nephrotoxicity,neurotoxicity, ototoxicity, myelosuppression, cardiotoxicity, alopecia,infertility and local inflammation from extravasation into the skin,xerostomia and mucositis.

The methods of the present invention are also suitable for treatingcomparable toxicities associated with anti-virals such as ddI(didanosine), ddC (zalcitabine), d4T (stavadine), 3TC (lamivudine), AZT(zidovudine, 3′azido-3′-deoxythymidine) and the like, anti-bacterialssuch as aminoglycosides, and anti-fungals, such as amphotericin B.

5.3.2 Nephro-disorders to be Treated

The following nephro-disorders, which are also referred to herein asrenal diseases, may be treated according to the methods of the presentinvention: Various types of glornerulonephritis, including diffuse formsof glornerulonephritis such as acute poststreptococcal, acutenonstreptococcal, rapidly progressive, chronic progressive, andend-stage chronic; focal forms of glomerulonephritis, such as those withsystematic bacterial infection, of probable immunologic origin, i.e.,IgA focal glomerulonephritis, and hereditary forms; nephrotic syndromessuch as minimal change disease, lipoid nephrosis, or nil disease, focalsegmental glomerular sclerosis, congenital nephrotic syndrome,membranoproliferative glomerulonephritis, idiopathic membranousnephropathy (membranous glomerulonephritis), systemic lupuserythematosus, systemic infection or hypersensitivity reactions,circulatory disturbances and renal vein thrombosis, amyloidosis, andtoxemia of pregnancy; renal diseases of vascular origin such ashypertensive vascular disease, benign nephrosclerosis, malignantnephrosclerosis, diabetic nephropathy, renal infarction, polyarteritisnodosa and Wegener's granulomatosis; thrombotic renal diseases such asdisseminated intravascular coagulation, bilateral renal corticalnecrosis, hemolytic uremic syndrome, and thrombotic thrombocytopenicpurpura; scleroderma; radiation nephritis; tubular diseases such asacute tubular necrosis, including toxic nephropathy and ischemic tubularnecrosis, osmotic nephrosis, hypokalemic nephropathy, chronicinterstitial and tubular diseases such as interstitial nephritis,pyelonephritis, tuberculous pyelonephritis, urinary tract obstructivedisease, renal papillary necrosis, analgesic abuse nephropathy, multiplemyeloma nephropathy, gout nephropathy, hypercalemic nephropathy andrenal calcinosis, and renal lithiasis; congenital malformations andanomalies such as agenesis and hypoplasia, fusion, ectopia, andreduplication, dysplasia and polycystic dysplasia, congenitalobstructive microcystic disease, simple cysts, infantile polycysticdisease, adult polycystic disease, and medullary cystic disease; renalneoplasms such as benign tumors, which include adrenocortical nodules,hamartomas, mesenchymal tumors, and cortical tubular adenomas; andmalignant tumors, such as adenocarcinoma, Wilms' tumor, leukemicinfiltration, and transitional cell carcinoma.

5.3.3 Metabolic Disorders to be Treated

The present invention provides methods for the treatment of a widevariety of metabolic disorders. One such metabolic disorder is diabetes.The methods of the present invention are also suitable for the treatmentof disorders relating to basal metabolism, i.e., heat production of anindividual at the lowest level of cell chemistry in the waking state, orthe minimal amount of cell activity associated with the continuousorganic functions of respiration, circulation and secretion;carbohydrate metabolism, i.e., the changes that carbohydrates undergo inthe tissues, including oxidation, breakdown, and synthesis; electrolytemetabolism, i.e., the changes which the various essential minerals,sodium, potassium, calcium magnesium, etc. undergo in the fluids andtissues of the body; fat metabolism, i.e., the chemical changes,oxidation, decomposition, and synthesis, that fats undergo in thetissues; protein metabolism, i.e., the chemical changes, decompositions,and synthesis that protein undergoes in the tissues; and respiratorymetabolism, i.e., the exchange of respiratory gases in the lungs and theoxidation of foodstuffs in the tissues with the production of carbondioxide and water.

5.3.4 Disorders Associated with Diabetes

Diabetes patients often suffer from numerous debilitating disorders. Onesuch disorder, peripheral neuropathy, is particularly likely to occur inthe older diabetic patient, with approximately 30% to 50% of thepatients showing minor reflex changes and evanescent pains in theextremities. The basic pathologic change in the peripheral nerves is asegmental demyelination. The autonomic nervous system may also beinvolved in diabetic patients, with resultant development of severediarrhea and abdominal pain. Greatly elevated levels of sorbitol andfructose have been demonstrated in peripheral nerves of animals withexperimentally induced diabetes. The accumulation of sorbitol andfructose is apparently attributable to a partial shunting of themetabolism of glucose through the aldose reductase pathway. It isunknown whether this abnormal metabolism of glucose with the formationof sorbitol is responsible for the decreased nerve conduction andsegmental demyelination in diabetic subjects. In experimental diabetes,degenerative changes have been found in autonomic nerve fibers of theintestinal tract of rats and were associated with the development ofmegacolon in these animals. Control of the diabetes by islettransplantation resulted in either prevent or disappearance of thedegenerative lesions in the autonomic nerves.

Kidney disease is common in diabetes and renal failure is one of themajor causes of death. The dominant form of diabetic nephropathy ismicrovascular disease affecting the renal glomerulus. Early in diabetes,the kidney increases in size and the associated glomerular hypertrophyleads to an increased glomerular filtration rate with hyperfiltrationand microalbuminuria in up to 50% of patients with new onset insulindependent diabetes mellitus. Later in the disease, diffuse thickening ofthe glomerular basement membrane is noted along with increased mesangialvolume and progressive impairment of renal function which in turn leadsto further expansion of the mesangium and eventual glomerular occlusion.

Clinically, in some cases mild proteinuria can remain constant for manyyears, while in other cases it progresses to reduction in glomerularfiltration and renal function with all the classical features ofnephrotic syndrome. Once azotemia (increased serum creatinine and BUN)develops, progression to renal failure and uremia is inevitable within afew months to two to three years. Once renal failure develops, the onlyalternatives are dialysis or transplantation.

The treatment of the above disorders is included within the scope of thepresent invention.

5.4 Effective Dosages

Pharmaceutical compositions suitable for use with the present inventioninclude compositions wherein the active ingredient is contained in atherapeutically effective amount, i.e., an amount effective to achieveits intended purpose. Of course, the actual amount of active ingredientwill depend on, among other things, the particular disorder beingtreated. Determination of an effective amount is well within thecapabilities of those skilled in the art.

For any compound described herein the therapeutically effective amountcan be initially estimated from cell culture assays. For example, a dosecan be formulated in animal models to achieve a circulatingconcentration range of compound, and/or an active metabolite thereof,that includes an effective concentration as determined in cell culture.Such information can be used to more accurately determine useful dosesin humans. See, e.g., Washburn et al., 1976, “Prediction of theEffective Radioprotective Dose of WR-2721 in Humans Through anInterspecies Tissue Distribution Study” Radiat. Res. 66:100-5.

Therapeutically effective amounts for use in humans can also beestimated from animal models. For example, a dose for humans can beformulated to achieve a circulating concentration found to be effectivein animals.

A therapeutically effective dose can also be estimated from currentclinical experience and data, including human pharmacokinetic data.While not intending to be bound by any particular theory, it is believedthat efficacy is related to a subject's total exposure to an applieddose of administered drug, and/or an active metabolite thereof, asdetermined by measuring the area under the blood concentration-timecurve (AUC). Thus, a dose administered according to the methods of theinvention that has an AUC of administered compound (and/or an activemetabolite thereof) within about 50% of the AUC of a dose known to beeffective for the indication being treated is expected to be effective.A dose that has an AUC of administered compound (and/or an activemetabolite thereof) within about 70%, 80% or even 90% or more of the AUCof a known effective dose is preferred. Adjusting the dose to achievemaximal efficacy in humans based on the methods described above,particularly on the blood concentration and duration of administeredcompound and/or its active metabolites is well within the capabilitiesof the ordinarily skilled artisan.

Usual patient doses for administration of amifostine and/or its activemetabolite WR-1065 usually range from about 50 mg/day to 6000 mg/day,commonly from about 100 mg/day to 4000 mg/day, and typically from about200 mg/day to 3500 mg/day. Stated in terms of patient body weight, usualdosages range from about 0.6 to 100 mg/kg/day, commonly from about 1.1to 66 mg/kg/day, and typically from about 2.2 to 58 mg/kg/day. Stated interms of patient body surface areas, usual dosages range from about 23to 4000 mg/m²/day, commonly from about 45 to 2666 mg/m²/day, andtypically from about 90 to 2333 mg/m²/day.

For other modes of administration, dosage amount and interval can beadjusted individually to provide effective plasma and/or tissue levelsof the administered compound, and/or an active metabolite thereof,according to the pharmacokinetic profiles described herein, aspreviously described.

The actual amount of composition administered will, of course, bedependent on the subject being treated, the subject's weight, theseverity of the affliction, the mode of administration and the judgementof the prescribing physician.

Dosages are in the range of between about 10-1000 mg/m² administeredparenterally. Preferred doses for intravenous administration are betweenabout 100-750 mg per m² of body surface area, more preferably betweenabout 200-750 mg/m². Preferred doses for oral administration are betweenabout 20-2000 mg per m² of body surface area, more preferably betweenabout 500-1500 mg/m² body surface area.

5.5 Formulations and Dosage Administration

The aminothiol compounds described herein, or pharmaceuticallyacceptable addition salts or hydrates thereof, can be delivered to apatient according to the invention using a wide variety of routes ormodes of administration. Suitable routes of administration include butare not limited to, inhalation, or parenteral routes, includingintravenous (infusion or bolus injection), intramuscular,intraperitoneal, intrathecal, subcutaneous, intranasal, transmucosal,buccal, sublingual, vaginal, rectal, intestinal, local intradermal ortransdermal routes. Alternatively, or concurrently, administration maybe by the oral route. Intravenous administration is particularlydesirable.

The aminothiol compounds described herein, or pharmaceuticallyacceptable salts and/or hydrates thereof, or mixtures thereof, may beadministered alone, or in combination with other aminothiol compounds ofthe invention, and/or in combination with one or more therapeuticagents, including cancer chemotherapeutic agents, intended to also treatthe toxicity or disorder suffered by the subject being treated. Examplesof such additional drugs include but are not limited to vitamins, inparticular the B complex.

Medicaments are considered to be provided “in combination” with oneanother if they are provided to the subject concurrently, sequentiallyor if the time between the administration of each medicament is such asto permit an overlap of biological activity.

The aminothiol compounds of the present invention may be administered byany means that achieve their intended purpose. Amounts and regimens forthe administration of the aminothiol rescue agents can be determinedreadily by those with ordinary skill in the clinical art of treatingneuro- and nephro-disorders, toxicities or cancer.

It is understood that the dosage of the aminothiol compound will bedependent upon the age, sex, health, and weight of the recipient, kindof concurrent treatment, if any, frequency of treatment, and the natureof the effect desired. An effective amount of the active compound of thepresent invention is any amount which would serve to treat or reversesymptoms of the neuro- or nephro-disorder or the neurotoxicity ornephrotoxicity caused by administration of a chemotherapeutic agent invivo. The ranges of effective doses provided herein are not intended tolimit the invention and represent preferred dose ranges. However, themost preferred dosage will be tailored to the individual subject, as isunderstood and determinable by one of ordinary skill in the art withoutundue experimentation.

For any mode of administration, the actual amount of compound delivered,as well as the dosing schedule necessary to achieve the advantageouseffects described herein, will also depend, in part, on such factors asthe bioavailability of the compound (and/or an active metabolitethereof), the disorder being treated, the desired therapeutic dose, andother factors that will be apparent to those of skill in the art. Theactual amount delivered and dosing schedule can be readily determined bythose of skill without undue experimentation by monitoring the bloodplasma levels of administered compound and/or an active metabolitethereof, and adjusting the dosage or dosing schedule as necessary toachieve the desired therapeutic effect. Additionally the dosage ordosing schedule can be adjusted as necessary to achieve the desiredtherapeutic effect by monitoring the signs and symptoms of the disorder.

The active compound(s) may be administered alone or in the form of apharmaceutical composition, wherein the active compound(s) is inadmixture with one or more pharmaceutically acceptable carriers,excipients or diluents. Pharmaceutical compositions for use inaccordance with the present invention may be formulated in conventionalmanner using one or more physiologically acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Properformulation is dependent upon the route of administration chosen.

The present methods also include providing a liquid based dosage form ofthe active compound suitable for administration to a subject in needthereof. The liquid base may be any liquid capable of transporting theactive ingredient into the body without disrupting the activity of thecompound or harming the patient. A preferred base is an isotonicsolution, which may also contain conventional additives such as sugars.These solutions are useful for both oral and intravenous administration.

For injection, the agents of the invention may be formulated in aqueoussolutions, preferably in physiologically compatible buffers such asHanks's solution, Ringer's solution, or physiological saline. Fortransmucosal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art.

Suitable injectable solutions include intravenous subcutaneous andintramuscular injectable solutions. The active compound may also beadministered in the form of an infusion solution or as a nasalinhalation or spray.

For intravenous administration, the active compound is preferablyadministered by drip infusion in an aqueous solution. The activeingredient may be administered in single or divided doses.

Suitable formulations for parenteral administration include aqueoussolutions of the active compounds in water-soluble form, for example,water-soluble salts. In addition, suspensions of the active compounds asappropriate oily injection suspensions may be administered. Suitablelipophilic solvents or vehicles include fatty oils, for example, sesameoil, or synthetic fatty acid esters, for example, ethyl oleate ortriglycerides. Aqueous injection suspensions that may contain substanceswhich increase the viscosity of the suspension include, for example,sodium carboxymethyl cellulose, sorbitol, and/or dextran. Optionally,the suspension may also contain stabilizers.

For oral administration, the active ingredient, preferably amifostine ora functional derivative or analogue thereof such as WR-151327, may be apreparation in any dosage form capable of oral administration. Suchdosage forms include tablets, hard or soft gelatin capsules, caplets,dragees, pills, tablets including coated tablets, and solutionsincluding elixirs, suspensions, gels, slurries or syrups, and the like.Pharmaceutical preparations for oral use can be obtained solidexcipient, optionally grinding a resulting mixture, and processing themixture of granules, after adding suitable auxiliaries, if desired, toobtain tablets or dragee cores.

The active compound of the present invention may be administeredrectally in the form of suppositories or enemas.

In general, the preparation in which the active compound of the presentinvention is administered contains from about 0.1 to about 100 percent,preferably from about 25-85 percent, of active compound(s), togetherwith a carrier or excipient. Suitable pharmaceutically acceptablecarriers comprise excipients and auxiliaries which facilitate processingof the active compounds into preparations which can be usedpharmaceutically. Suitable excipients are, in particular, fillers suchas sugars, such as lactose, sucrose, mannitol, or sorbitol; cellulosepreparations and/or calcium phosphates, such as tricalcium phosphate orcalcium hydrogen phosphate; as well as binders such as starch paste madeusing, for example, maize starch, wheat starch, rice starch, potatostarch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethylcellulose, sodium carboxymethyl cellulose, and/orpolyvinylpyrrolidone. If desired, disintegrating agents may also beadded, such as the above-mentioned starches as well as carboxymethylstarch, cross-linked polyvinyl pyrrolidone, agar, or alginic acid or asalt thereof, such as sodium alginate. Auxiliaries which can be used inthe compositions according to the present invention includeflow-regulating agents and lubricants such as silica, talc, stearic acidor salts thereof, and/or polyethylene glycol.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for suchadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g. gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation, subcutaneous or intramuscularinjection. Thus, for example, the compounds may be formulated withsuitable polymeric or hydrophobic materials (for example as an emulsionin an acceptable oil) or ion exchange resins, or as sparingly solublederivatives, for example, as a sparingly soluble salt.

The pharmaceutical compositions also may comprise suitable solid or gelphase carriers or excipients. Examples of such carriers or excipientsinclude but are not limited to calcium carbonate, calcium phosphate,various sugars, starches, cellulose derivatives, gelatin, and polymerssuch as polyethylene glycols.

It will be understood that the aminothiol rescue agents of the inventionmay be administered in accordance with the methods of the invention atany time during or after administration of the chemotherapeutic agent,preferably after administration. For example, the rescue agent may beadministered one hour after, or more preferably, four or more hoursafter administration of the chemotherapeutic agent. Most preferably, therescue agent is administered days, or even weeks after chemotherapy. Therescue agent may be administered after toxicities associated withadministration of chemotherapy are observed. Preferably, the rescueagent is administered after chemotherapeutically-induced toxicities areobserved or demonstrated.

Indications of toxicity in a patient who has received chemotherapy arewell known to those of ordinary skill in the art of treating cancerpatients. For example, indications or “markers” of neurotoxicity causedby chemotherapy include, but are not limited to clinical signs andsymptoms, nerve conduction studies, and vibrometer measurements. Markersof nephrotoxicity caused by chemotherapy include, but are not limited toincreased levels of serum creatinine above 1.5 mg/Dl, BUN greater than20, abnormal electrolytes, for example decrease in serum, magnesium,bicarbonate or elevations in serum potassium. Preferably, the aminothiolcompounds of the invention are administered after one or more of suchtoxicity markers are observed following chemotherapeutic treatment.

It will be understood that the aminothiol compounds of the invention maybe administered according to the methods of the invention after any oneor more cycles of administration of a chemotherapeutic drug. Forexample, the rescue agent may be administered after one cycle ofadministration of a chemotherapeutic agent, but prior to the next cycleof administration of chemotherapeutic agent.

Manufacturing and packaging amifostine comprises the steps of fillinginto pre-sterlized vials a sterilized water solution comprisingamifostine to a predetermined volume, cooling the vials and theircontents, and removing the solvent by lyophilization to produce driedamifostine of a predetermined amount in each vial. [See L. Lachman, etal. The Theory and Practice of Industrial Pharmacy p 62-63, 1986].

Unexpectedly, it has been discovered that a sterile and stable productof crystalline amifostine with and without excipient(s) such as mannitolcan be prepared from the vacuum drying of an amifostine drugsubstance-containing hydro-ethanolic solution of about 1 to about 35%ethanol.

An important aspect of the present invention involved preformulationstudies that determined (1) the solubility of amifostine drug substance(mg/ml) at various concentrations of water/ethanol, (2) the solubilityof amifostine drug substance in −25 water/ethanol at varioustemperatures, (3) the appropriate shelf temperature of the freeze-dryerneeded to effect precipitation of amifostine before vacuum drying and(4) the concentration of ethanol needed in the formulation to give asuper-saturated solution that when cooled to the desired shelftemperature in the freeze-dryer results in the precipitation ofamifostine in a crystalline form. From the above preformulation studies(see Examples, infra) it was determined that the preferred concentrationof amifostine, on an anhydrous basis, was about 100 mg/ml in about 10%aqueous ethanol. Further, a preferred shelf temperature of about −20° C.would effect precipitation of amifostine.

In order to obtain an elegant cake product, the percentage of ethanol inthe ethanol/water mixture ranges from about 1 to about 35% v/v ofethanol (e.g., ratio of ethanol:water 1:99; 35:65); similarly, the shelftemperature of the freeze-dryer can range from about −40° C. to about−10° C., preferably −20° C. The results of the preformulation studiespresented in this invention provide an important basis for adjustment ofthe interdependent variables of amifostine concentration, ethanolconcentration and temperature to provide for multiple containersize/fill volume combinations.

Generally, the freeze-dryer shelf is pre-chilled to a temperature ofabout −30° C. to about −15° C., preferably about −20° C. The vials areloaded and the temperature is readjusted to about −30° C. to about −15°C. and preferably −20° C. and the vials are maintained at thistemperature for about 20 hours. Depending on the concentration ofethanol and amifostine or amifostine and excipient in the solutions, anddepending on ethanol concentration, the temperature necessary to effectprecipitation will vary accordingly. Next, the precipitation ofcrystalline amifostine takes place, followed by the freezing of theformulation.

Once the frozen formulation is observed, the primary drying cycle isinitiated to remove bulk water and ethanol. Generally, the pressure inthe chamber is reduced to about 150 mTorr. The primary drying cycle iscomplete when the formulation temperature was approximately −20°+−2° C.for more than two hours. During the secondary drying process, theformulation is held at about −20° C. to about 10° C., preferably at atemperature above the primary drying cycle temperature, for about 40 toabout 50 hours to facilitate secondary drying, i.e. removal of residualwater and ethanol. When the partial pressures of water and ethanol inthe chamber reaches a steady state, the drying is considered to becompleted. These formulations provide a vacuum dried product which hasbeen found to be a crystalline amifostine that demonstrates improvedstability over the current formulation which contains amorphousamifostine. The vials can then be stored and shipped at temperaturesfrom about 4° C. to about room temperature without significant productdegradation.

Moreover, excipients can be added to increase the amount of solidspresent in the formulation. Among the excipients found useful for thispurpose, often in combination, are sodium or potassium phosphates,sodium chloride, citric acid, tartaric acid, gelatin and carbohydratessuch as dextrose, sucrose, sorbitol, inositol, mannitol and dextran. Inaddition to those mentioned herein others are known to those skilled inthe art.

The vacuum dried crystalline amifostine solid compositions of thepresent invention may be provided in single dose container forms byaseptically filling suitable containers with the sterile pre-vacuumdried solution to a prescribed amifostine content; preparing the desiredvacuum dried solid composition; and then hermetically sealing the singledose container. It is intended that these filled containers will allowrapid dissolution of the solid composition upon reconstitution withappropriate sterile diluents in situ giving an appropriate sterilesolution of desired amifostine concentration for administration. As usedherein, the term “suitable containers” means a container capable ofmaintaining a sterile environment, such as a vial, capable of deliveringa vacuum dried product hermetically sealed by a stopper means.Additionally, suitable containers implies appropriateness of size,considering the volume of solution to be held upon reconstitution of thevacuum dried composition; and appropriateness of container material,generally Type I glass. The stopper means employed, e.g., sterile rubberclosures or an equivalent, should be understood to be that whichprovides the aforementioned seal, but which also allows entry for thepurpose of introduction of diluent, e.g., sterile Water for Injection,USP, Normal Saline, USP, or 5% Dextrose in Water, USP, for thereconstitution of the desired amifostine solution. These and otheraspects of the suitability of containers for pharmaceutical productssuch as those of the instant invention are well known to those skilledin the practice of pharmaceutical arts.

While the physical properties, such as appearance, were improved in theinstant solid compositions, thereby achieving one objective of theinvention, we unexpectedly found that these instant solid compositionsalso possessed improved thermal stability compared with currently knownformulation. In practice, expectation for enhancement of chemicalstability by vacuum drying relates to a comparison of the stability ofthe vacuum dried solid with the stability of the solution form of thepharmaceutical composition. In contrast, the instant compositionsdemonstrate enhanced chemical stability between solid dosage forms, seeExamples infra.

The pharmaceutical compositions of the present invention are suitablefor parenteral administration, for example, intravenous, intramuscular,intracavitary, intrathecal, and subcutaneous injections.

In a specific embodiment of the present invention, the process includesa sterilization step. Sterilization may be effected, for example, bysterile filtering a solution, e.g., through a 0.2 μm pore size filter.It should be noted further that the crystalline aminoalkyl dihydrogenphosphorothioate may be anhydrous or contain solvents ofcrystallization. In particular, crystalline amifostine may be anhydrous,a solvate, or a hydrate, such as a monohydrate or a trihydrate.Generally, the hydrates may contain about 1 to about 5, preferably about1 to about 3, moles of water of crystallization.

Having now generally described the invention, the same will be morereadily understood through reference to the following examples which areprovided by way of illustration, and are not intended to be limiting ofthe present invention, unless specified.

6. WORKING EXAMPLES 6.1. Example 1 Case Study

A 61-year-old patient with new onset cisplatin-induced renal failurethat was identified 6 days post-cisplatin therapy was treated withamifostine 400 mg intravenously for 3 days, with partial recovery of herrenal function and improvement in her overall clinical status.

The patient was referred by her primary care physician for a gynecologicevaluation for complaints of increasing pain in her lower abdomen withcostovertebral angle tenderness over a 10-month period. Ovarian cystswere identified and monitored by ultrasound on several occasions duringthat time. The patient had a history of recurrent urinary tractinfections, and she had a stent placed for approximately two weeks priorto the gynecologic consult. A CAT scan at that time revealed ureteralstenosis and slight hydronephrosis of her right kidney. Other relevantpast medical history included congenital absence of her left kidney,double uteri, and a total abdominal hysterectomy and leftsalpingo-oophorectomy approximately 30 years previously.

The patient underwent an exploratory laparotomy to determine if theetiology of her increasing pain was secondary to the cystic ovary,urinary tract infections, or ureteral stenosis and hydronephrosis. Thepatient was found to have grade III papillary adenocarcinoma arisingfrom a serious type adenofibroma, with metastasis to the omentum. Aright oophorectomy and partial omentectomy were performed, with lysis ofadhesions. Repeat surgery was performed approximately one month laterfor lymph node biopsy for staging of the cancer and to remove additionalomentum. Intraperitoneal and intravenous (iv) catheters were placed atthis time for future chemotherapy.

The patient was evaluated for chemotherapy and was placed on a treatmentregiment consisting of paclitaxel 135 mg/m² (223 mg) administered iv onday 1, followed by cisplatin 100 mg/m² (165 mg) intraperitoneally on day2. However, after receiving days 1 and 2 of chemotherapy, the patientpresented to the emergency room on day 3 with nausea, vomiting, andabdominal cramping. She was admitted for hydration and treatment ofnausea. For the next 5 days, she continued to complain of nausea andvomiting, despite treatment with intensive antiemetic regimens,including granisetron, dexamethasone, and promethazine. Other treatmentat that time included ketorolac for pain, lorazepam for anxiety, andlarge volumes of iv fluids. By hospital day 5, the patient's serumcreatinine had risen to 4.9 mg/Dl and the BUN to 39 mg/Dl. Electrolyteabnormalities at that time were: sodium—128 mMol/L, potassium—1.9Mmol/L, chloride—94 Mmol/L, and CO₂—22 Mmol/L. Also, the serum calciumwas 4.5 mMol/L. The patient was transferred to telemetry for observationand normalization of her electrolytes.

A renal consultation determined the patient's acute renal failure to besecondary to cisplatin therapy and complicated by volume depletion. Shecontinued to be rehydrated, and further chemotherapy was withheld.Because of persistent, unresponsive renal failure, amifostine 400 mg perday was administered intravenously on post-cisplatin days 7, 8, and 9 inan effort to reduce the drug-induced nephrotoxicity. The patienttolerated therapy well, with only mild nausea. Her renal functionimproved over the next 3 days on amifostine, with her creatininedecreasing from a peak concentration of 6.0 mg/dL before amifostine to4.6 mg/dL on day 3 of amifostine therapy. At that time, her electrolyteshad normalized, her abdominal cramping significantly lessened, and shewas able to tolerate oral fluids. By hospital day 12, the patient'screatinine had decreased to 2.8 mg/dL and she was discharged on hospitalday 13. The patient's response to amifostine therapy, as demonstrated byimprovement in serum creatinine, is shown in FIG. 1.

Two days later the patient was readmitted with dystonia caused byincreased promethazine usage for intractable nausea and vomiting. Thepatient was hydrated and monitored over the next 5 days, and electrolyteabnormalities were corrected. Amifostine 400 mg iv was administered onceagain on hospital day 2, with ondansetron 24 mg administered as neededfor nausea. The patient tolerated the treatment well. Her renal functionstabilized during this hospitalization, with creatinine/BUN rangingbetween 2.8/17 mg/dL and 3.2/13 mg/dL. She was discharged on hospitalday 5.

A follow-up clinic visit was scheduled for 3 days post-discharge, atwhich point the patient chose not to pursue further chemotherapy.Amifostine 400 mg iv was administered 5 days later to assess whetherfurther improvement in her renal function could be achieved. Granisetron1 mg was given orally as premedication. The patient's creatinine at thattime was 2.5 mg/dL. Two weeks later, it was decided to administer afinal 2-week course of amifostine 400 mg, with reassessment at the endof therapy. Amifostine was administered every 2 to 3 days for a total of5 doses over the 2 weeks. Lorazepam 1 mg iv and ondansetron 24 mg ivwere administered as premedication. Approximately 6 months later, thepatient's serum creatinine was 2.0 mg/dL.

In this Example, the patient received amifostine on days 7 through 9post-cisplatin therapy, after renal failure had already developed.Amifostine partially reversed the toxic renal effects of cisplatin, withthe patient's serum creatinine decreasing over the 3-day course oftreatment from 6.0 mg/dL to 4.6 mg/dL, and to 2.8 mg/dL by dischargefrom the hospital on day 13. Concurrent with her improving renalfunction, the patient's overall clinical status improved during thistime, with normalization of electrolyte imbalances and defervescence ofabdominal cramping, nausea, and vomiting.

6.2. Example 2 Case Study

The patient, a 75-year-old white male, who is a survivor of theconcentration camps of World War II, was diagnosed with bladder cancerthat had metastized to the lungs. He received 3 cycles carboplatinum,vinblastine and methotrexate. While in the concentration camp, thepatient suffered damage to his lower legs which caused him constantpain. This pain was made worse by chemotherapy. The tumors wereshrinking in his lungs, but he developed severe neurotoxicities of hisright arm and refused further treatment. These neurotoxicities seemed toworsen after chemotherapy was stopped. He was given demerol for acuterelief and required 2-100 mcg/hr. Duragesic Patches in order to make hispain “bearable.” The patient was tried on many other agents andcombination of agents including steroids and non-steroidals. Soon theDuragesic patches were no longer making his pain bearable, and he wasvery depressed, and complaining of sleepiness.

After premedication with antiemetics, the patient was given 500 mg ofamifostine in 50 cc 0.9 saline over 10 minutes. Two days later, thepatient reported that he had no pain for the first time in months, eventhe pain in his legs was getting better. He was able to walk without hiscane. The patient received subsequent Ethyol infusions and has remainedwithout arm pain. The pain in his legs has improved and he is managingwith only one Duragesic Patch.

6.3. Example 3 Case Study

A 72-year-old white female was diagnosed with lung cancer and received 3cycles of amifostine, paclitaxel, and carboplatin in which theamifostine was administered prior to chemotherapy. By the fourth cycleshe was confined to a wheel chair with foot drop and unable to feedherself or brush her teeth because of her neurotoxicities. Chemotherapyhad to be stopped even though her tumor was responding to treatment.Several medications were tried along with physical therapy to reduce herproblems of neuropathy. When she did not respond to several months ofthis treatment, Ethyol single agent therapy was initiated.

The patient was premedicated with Zofran (ondanestron) 32 mg, Decadron20 mg in 50 cc normal saline IVPB over 15 minutes and then givenamifostine 970 mg in 50 cc normal saline over 10 minutes. She toleratedthis treatment without problems. Two days later, the patient reportedimprovement in strength in her upper arms and legs. She was able to holda tooth brush and silverware again. The patient continued to receiveweekly infusions of amifostine. By the third treatment, she was walkingagain with the aid of a walker. However, the day after the fourthtreatment with amifostine, the patient experienced erythema andblistering all over her body, which eventually led to total skinpeeling. The amifostine treatments were terminated. One month later, thepatient's skin was back to normal and her neuropathy remained stable.

6.4 Example 4 Case Study

A 55-year-old male with myelodysplastic bone marrow syndrome (MDS)suffered with neurotoxicities for eight years which developed as aresult of CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone)chemotherapy given for Non-Hodgkins Lymphoma. This patient initiatedtreatment for MDS with Ethyol, 840 mg, Monday, Wednesday and Friday ofeach week. After receiving five weeks of such amifostine therapy forMDS, the patient stated (unsolicited) that his neurotoxicities werebetter since receiving amifostine. The patient continues on Ethyol forMDS and is doing well.

6.5 Example 5 Method of Producing Crystalline Amifostine withoutMannitol

To 130 mL of water at 25° C., add with stirring 21.25 gm of amifostinedrug substance trihydrate, which is equivalent to 17.0 gm of anhydrousamifostine drug substance. After dissolution of amifostine drugsubstance is complete, 17 mL absolute ethanol, USP, is added to thesolution with stirring. Water is then added to QS 170 mL. The resultingsolution is sterile filtered through a 0.22 μm filter. To each ofthirty-three 10 mL vials, is dispensed 5 mL of the filtered solution togive 500 mg amifostine, on an anhydrous basis, per vial in anethanol:water ratio of 10:90. Split stoppers are placed on the vials andthe samples are subjected to the following vacuum drying cycle: thesamples are placed on the shelves of the freeze dryer, which has beenpre-cooled to about −20° C., for about 17 hours at ambient pressure,after which time the chamber is evacuated and the shelves are held atabout −20° C. for 28 hours. Following this period, the chamber isback-filled with nitrogen and the vials are quickly stoppered by hand.This procedure results in a thermally-stable, freeze-dried single dosevial containing approximately 500 mg of crystalline amifostine as anelegant cake.

6.6 Example 6 Method of Producing Crystalline Amifostine

Approximately 20 grams of mannitol is added with stirring to 150 mL ofwater at 25° C. To this solution is added, with stirring, approximately25 grams amifostine drug substance (trihydrate basis), which isequivalent to 20 grams of anhydrous amifostine drug substance. Afterdissolution is complete, 20 mL of anhydrous ethanol, USP, is addedvolumetrically to the solution with stirring. Water is added QS to 200mL. The resulting solution is sterile filtered through a 0.2 μm filterand 5 mL of solution is transferred to each of 40 10-mL vials. Splitstoppers are placed on the vials and the samples are loaded onto thefreeze-dryer shelf at ambient temperatures. The shelf temperature isdecreased at 2° C./min to −25° C. and held at this temperature for 90minutes to initiate amifostine crystallization. After this time, theshelf temperature is raised above the eutectic point at a rate of 2°C./min to −5° C. and held at this temperature for 10 hours to anneal theproduct. Subsequently, the shelf temperature is lowered to −25° C. untilthe product temperature is less than −18° C. for greater than 60minutes. After this time, the freeze-dryer condenser is turned on andthe vacuum in the chamber is lowered to 150 mTorr. The shelf-temperatureis raised to −20° C. and the samples are allowed to vacuum dry for 14hours. At this point, the monitored vials have reached shelftemperature, indicating the end of the primary drying cycle. The vialsremain at 150 mTorr on the −20° C. shelf for an additional 13.4 hours toensure the removal of non-hydrate water. The chamber is back-filled withnitrogen and the vials are mechanically stoppered. This procedureresults in a thermally-stable, vacuum-dried single dose vial containingapproximately 500 mg of amifostine (anhydrous basis) and 500 mg mannitolas an elegant cake.

6.7 Example 7 Vacuum Dried Crystalline Amifostine Stability Testing

Several sealed, nitrogen-filled vials of crystalline amifostineformulated from 10:90 ethanol:water, as described in Example 5 above,are stressed at 50° C. for up to 35 days to determine the thermalstability of the crystalline amifostine.

The results are tabulated in Table 1 below. All data are reported aspercent (%) of initial concentration, which is defined as 100%. TABLE 1TIME AT 50° C. % OF STUDY (IN DAYS) INITIAL CONCENTRATION 1 0 100.0 3106.3 35 96.9 2 0 100 3 97.2 7 101.1 14 93.9 21 71.1 3 0 100 3 103.6 7101.8 14 97.5

For comparison purposes, the current amorphous amifostine formulation isalso subjected to stress testing at 50° C. for up to 28 days. Theresults are presented in Table 2 below. All data are reported as percent(%) of initial concentration, which is defined as 100%. TABLE 2 % OFTIME AT 50° C INITIAL STUDY (IN DAYS) CONCENTRATION 1 0 100.0 14 2.8 281.5 2 0 100 14 2.0 28 1.4 3 0 100 14 1.7 28 1.4

Hence, it is abundantly clear that, even between solid formulations, adramatic increase in thermal stability is achieved by crystallinecompositions obtained from the disclosed process.

6.8 Example 8 Preferred Method of Producing Crystalline Amifostine

Compounding Procedure for Amifostine/Mannitol (100 mg anydrous each/mL)

The following procedure was written to yield 3.5 liters of solution.

-   -   1. 350 grams mannitol (USP) were dissolved with stirring        (magnetic teflon stir bar) in about 2300 mL Nanopure water at        room temperature in a stainless steel pressure vessel.    -   2. 438.3 grams amifostine trihydrate was added to this solution.        Dissolution was aided with vigorous stirring.    -   3. After amifostine dissolution were complete, 525 mL dehydrated        ethanol (USP) was slowly added to the solution with vigorous        stirring. Amifostine precipitation occurs at the addition site        followed by rapid re-dissolution as the ethanol is diluted by        stirring.    -   4. After the addition of the ethanol is complete, the solution        was diluted to 3500 mL with Nanopure water.    -   5. The solution was filtered under a positive pressure of 10 psi        (nitrogen) through a Millipore-40 filter.    -   6. 5 mL of the resulting solution was transferred to each of 660        10-mL tubing vials (Wheaton E-2910-B47B). The vials were        partially seated with grey butyl rubber stoppers (Tompkins        PT23B0857 F2) and vacuum dried.

Vacuum drying Cycle for Amifostine/Mannitol (100 mg anhydrous/mL)

-   -   1. Vials are placed on the shelf at about 25° C. to insure that        amifostine precipitation is not initiated heterogeneously.    -   2. The shelf temperature is lowered at 2° C. per minute to        −35° C. Once this shelf temperature is obtained, it is held        constant for 240 minutes to insure solution freezing of all        vials. During this stage the samples pass through a eutectic        (approximately −16° C.).    -   3. At the end of the 240 minute hold time, the shelf temperature        is raised at 2° C. per minute to 0° C. over 25 minutes. Once        this shelf temperature is obtained, it is held constant for 600        minutes.    -   4. At the end of the 600 minute hold time, the shelf temperature        is again lowered to −35° C. at 2° C. per minute. Once this        temperature is obtained, it is held constant for 180 minutes.    -   5. After this time, the condenser is turned on. When the        condenser temperature is less than −40° C., the chamber is        evacuated. When the chamber pressure is less than 150 mT, the        shelf temperature is raised to −20° C. at 20° C. per minute and        the chamber pressure is held at 150 mT with a nitrogen chamber        bleed.    -   6. The product is left in the chamber at 150 mT for 12 to 24        hours after the monitored product temperature has reached shelf        temperature. The chamber is back-filled with nitrogen and the        vials stoppered.

NOTE: 1 Torr is equivalent to 1 millimeter of Hg at 0° C.

Sealed, nitrogen-filled 10 ml tubing vials containing vacuum driedcrystalline amifostine, obtained as described in Example 5, werestressed at 0° C. for 4 weeks. For crystalline amifostine dried at −20°C. for 12 hours, 93% of the amifostine remained at the end of the stresstest period. For cystalline amifostine dried at −20° C. for 24 hours,84% of the amifostine remained at the end of the stress test period.

6.9 Example 9 Most Preferred Method of Producing Crystalline Amifostine

It was found that the most stable vacuum dried, crystalline amifostinewas obtained by vacuum-drying an amifostine/mannitol, ethanol/watersolution containing 15% v/v ethanol. The compound procedure is the sameas described in Example 8 except for the lesser amount of dehydratedethanol added to the solution.

The specific manner of conducting the vacuum drying cycle to produce themost stable crystalline amifostine was arrived at after several studieswere performed to evaluate effects of changing the final dryingtemperature, the time period for final drying, and the rate of initialcooling to −35° C. of the solution-containing vials. It was found thatin general, the stability of crystalline amifostine is the greatest whenthe final drying temperature was at −20° C., and the time for the finaldrying was between 12 and 24 hours. Additionally, the stability of thecrystalline amifostine was higher when the initial cooling to −35° C. ofthe solution-containing vials was conducted in 160 minutes rather then45 minutes.

Based on the above development studies, the most preferred manner ofconducting the vacuum drying cycle is as follows:

Vacuum Drying Cycle for Amifostine/Mannitol (100 mg Anhydrous./mL)

-   -   1. Vials are placed on the shelf at about 25° C. to insure that        amifostine precipitation is not initiated heterogeneously.    -   2. The shelf temperature is lowered from 25° C. to 0° C. in 20        minutes, 0° to −20° C. in 60 minutes, and then from −20° C. to        −35° C. in 80 minutes. Once the shelf temperature is obtained,        it is held constant for 240 minutes to insure solution freezing        of all vials. During this stage the samples pass through a        eutectic (approximately −16° C.).    -   3. At the end of the 240 minute hold time, the shelf temperature        is raised to 0° C. over 25 minutes. Once the shelf temperature        of 0° C. is obtained, it is held constant for 600 minutes.    -   4. At the end of the 600 minute hold time, the shelf temperature        is again lowered from 0° C. to −15° C. in 15 minutes, and then        from −15° C. to −35° C. in 120 minutes. Once the temperature of        −35° C. is obtained, it is held constant for 180 minutes.    -   5. After this time, the condenser is turned on. When the        condenser temperature is less than −40° C., the chamber is        evacuated. When the chamber pressure is less than 150 mT, the        shelf temperature is raised from −35° C. to −20° C. at 2° C. per        minute while the chamber pressure is held at 150 mT with a        nitrogen chamber bleed.    -   6. The product in the vials is left in the chamber at 150 mT for        12 to 24 hours after the monitored product temperature has        reached shelf temperature. The chamber is back-filled with        nitrogen and the vials stoppered.

NOTE: 1 Torr is equivalent to 1 millimeter of Hg at 0° C.

Without wishing to be limited by theory, it is believed that above step2 causes the formation of seed crystals of amifostine in the frozensolution and step 3 causes the growth of amifostine crystals around theseed crystals and ensures completion of the crystallization ofamifostine from the partially frozen solution.

Crystalline amifostine has been produced using the above vacuum dryingcycle, utilizing 12.5% ethanol solution-one produced with a final dryingstep of 12 hours and another produced with a final drying step of 24hours. Stress testing of these two products at 40° C. for eight weeksindicate no perceptible decomposition of amifostine for the productdried for 12 hours, and a 2% decomposition of amifostine for the productdried for 24 hours.

6.10 Example 10 Preferred Manner of Conducting Crystalline AmifostineStability Testing

Sealed, nitrogen-filled 10 ml tubing vials containing vacuum driedcrystalline amifostine, obtained as described in Example 9, werestressed at 40° C. for up to eight weeks.

It was found that previous stability testing at 50° C. causeddecomposition of the crystalline amifostine in the sealed vials in amanner not easily correlated to the stability of the crystallineamifostine under typical storage conditions (i.e. at refrigerationtemperature of about 4° C.). However, results of stability testing at40° C. and less can be correlated to the stability of crystallineamifostine under typical storage conditions. As an approximation,stability for one month at 30° C. correlates to eighteen months at 4°C.; stability for 2-3 weeks at 40° C. correlates to 18 months at 4° C.;and stability for 8-12 weeks at 40° C. correlates to 18 months at 25° C.See L. Lachman, et al. The Theory and Practice of Industrial Pharmacypages 766-67 for a general discussion of stability prediction.

At the end of the stress period, the crystalline amifostine in the vialswas tested for water content, thiol content, and amifostine content. Thewater content was determined by Karl Fischer titration. Becauseamifostine may undergo hydrolysis under stress to produce2-[(3-aminopropyl)amino]ethane thiol and phosphoric acid, determinationof the amount of this thiol gives an indication of the stability of theamifostine. Analysis of thiol and amifostine content was conducted.

6.11 Example 11 Stability Results of Vacuum Dried, CrystallineAmifostine Stressed at 40° C.

Typical results obtained by stressing crystalline amifostine produced bythe method described in Example 9 and tested as described in Example 10are summarized in Table 3. TABLE 3 Stability Results of Vacuum DriedCrystalline Amifostine at 40° C. Time Acceptance % H₂O % Thiol %Amifostine Criteria 10-14% w/w NMT 2.0% w/w 38-46% w/w Initial Lot 81212.1 0.5 44.5 1 week 10.5 0.2 42.6 2 weeks 10.1 0.2 42.5 3 weeks 10.40.2 42.5 4 weeks 10.2 0.2 41.2 8 weeks 11.7 0.3 43.4 Lot 815 12.0 0.343.3 1 week 11.7 0.2 43.6 2 weeks 11.6 0.2 43.4 4 weeks 11.5 0.3 43.0NMT = no more than

The above results clearly indicate the enhanced stability of thecrystalline amifostine produced by the method described in Example 9.The enhanced stability is evident from the low weight percent of thiolformation, which indicates very little decomposition of the amifostineby hydrolysis to form 2-[(3-aminopropyl)amino]ethane thiol.Additionally, there is little loss in water content or amifostinecontent over time. This is in contrast to the poor stability of thevacuum dried amorphous amifostine formulation which exhibits significantdecomposition within 14 days at 50° C. (See Table 2 of Example 7).

6.12 Example 12 Crystal Structure of Vacuum Dried Amifostine

The molecular and crystal structure of vacuum dried crystallineamifostine has been determined. Crystal survey, unit cell determination,and data collection were performed using copper radiation at roomtemperature.

The structure was solved by direct methods and refined by full-matrixleast-squares and difference Fourier methods. All non-hydrogen atomswere refined anisotropically. The hydrogen atoms attached to thenitrogen and water oxygen atoms were located from difference Fouriermaps and refined isotropically. The positions of the remaining hydrogenatoms were calculated assuming ideal geometries. These hydrogen atomswere not refined due to the low reflection to parameter ratio.

The compound crystallizes in the chiral space group P2₁2₁2₁. The datapresented in this example are from the enantiomeric structure with lowerR values (R=0.036 and R_(w)=0.042). The other enantiomeric structure hasan R value of 0.042 and R_(w) value of 0.051. A graphic depiction of themolecular and crystal structure of vacuum dried amifostine trihydrate isshown in FIG. 2.

6.13. EXPERIMENTAL

Data Collection

A colorless flat needle-shaped crystal of C₅H₂₁N₂O₆PS having approximatedimensions of 0.350×0.050×0.030 mm was mounted on a glass fiber. Allmeasurements were made on a Rigaku AFC5R diffractometer with graphitemonochromated Cu Kα radiation and a 12 KW rotating anode generator.

Cell constants and an orientation matrix for data collection, obtainedfrom a least-squares refinement using the setting angles of 20 carefullycentered reflections in the range 40.45<2θ<52.03° corresponded to anorthorhombic cell with dimensions:a=8.456(2) Åb=21.553(2) Åc=6.758(2) ÅV=1231.6(5) Å³.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are intended to fall within the scope of the appendedclaims.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties.

1. A method for treating xerostomia associated with radiation therapyfor head and neck cancer in a human, which comprises administering tosaid human a therapeutically effective amount of a dosage formcomprising a thermally stable, sterile, crystalline compound of theformula:R₁NH(CH₂)_(n)NH(CH₂)_(m)SR₂ or a pharmaceutically acceptable additionsalt or hydrate thereof, wherein R₁ is hydrogen, C₅-C₇ aryl, C₂-C₇ acyl,or C₁-C₇ alkyl; R₂ is hydrogen, PO₃H₂ or R₃ wherein R₃ isR₁NH(CH₂)_(n)NH(CH₂)_(m)S—; n is an integer from 1 to 10; and m is aninteger from 1 to
 10. 2. The method of claim 1, wherein R₂ is PO₃H₂. 3.The method of claim 2, wherein R₁ is hydrogen.
 4. The method of claim 3,wherein n is 3 and m is
 2. 5. The method of claim 4, wherein thecompound is a thermally stable, sterile, crystalline amifostine hydrate.6. The method of claim 5, wherein the compound is thermally stable,sterile, crystalline amifostine trihydrate.
 7. The method of claim 6,wherein the crystalline amifostine trihydrate exhibits substantially thecrystal structure having a space group of P2₁2₁2, and cell dimensions ofabout a=8.46 Å, b=21.55 Å and c=6.76 Å.
 8. The method of claim 6,wherein the crystalline amifostine trihydrate forms less than about 2%2-[(3-aminopropyl)amino]ethane thiol when sealed in a nitrogen filledvial and heated to 40° C. for one week.
 9. The method of claim 6,wherein the crystalline amifostine trihydrate forms less than about 2%2-[(3-aminopropyl)amino]ethane thiol when sealed in a nitrogen filledvial and heated to 40° C. for four weeks.
 10. The method of claim 6,wherein the crystalline amifostine trihydrate is thermally stable atabout 4° C. for at least two years.
 11. The method of claim 6, whereinthe crystalline amifostine trihydrate is thermally stable at aboutambient temperature for at least two years.
 12. The method of claim 1,wherein the dosage form further comprises an excipient.
 13. The methodof claim 12, wherein the excipient is mannitol.
 14. The method of claim1, wherein the dosage form is lyophilized.
 15. The method of claim 6,wherein the dosage form comprises 500 mg of amifostine on an anhydrousbasis.
 16. The method of claim 1, wherein the amount of the crystallinecompound administrated is from about 10 to 1000 mg/m².
 17. The method ofclaim 1, wherein the amount of the crystalline compound administrated isfrom about 200 to 750 mg/m².
 18. The method of claim 1, wherein theamount of the crystalline compound administrated is about 750 mg/m². 19.The method of claim 1, wherein the dosage form is administrated beforethe radiation therapy.
 20. The method of claim 1, wherein the dosageform is administrated after the radiation therapy.
 21. The method ofclaim 1, wherein the dosage form is administrated during the radiationtherapy.
 22. A method for treating xerostomia associated with radiationtherapy for head and neck cancer in a human, which comprisesadministering to said human a therapeutically effective amount of adosage form comprising a thermally stable, sterile, crystallineamifostine.
 23. The method of claim 22, wherein the crystallineamifostine is an amifostine hydrate.
 24. The method of claim 23, whereinthe crystalline amifostine is an amifostine trihydrate.
 25. The methodof claim 24, wherein the crystalline amifostine trihydrate exhibitssubstantially the crystal structure having a space group of P2₁2₁2₁ andcell dimensions of about a=8.46 Å, b=21.55 Å and c=6.76 Å.
 26. Themethod of claim 25, wherein the crystalline amifostine trihydrate formsless than about 2% 2-[(3-aminopropyl)amino]ethane thiol when sealed in anitrogen filled vial and heated to 40° C. for one week.
 27. The methodof claim 25, wherein the crystalline amifostine trihydrate forms lessthan about 2% 2-[(3-aminopropyl)amino]ethane thiol when sealed in anitrogen filled vial and heated to 40° C. for four weeks.
 28. The methodof claim 25, wherein the crystalline amifostine trihydrate is thermallystable at about 4° C. for at least two years.
 29. The method of claim25, wherein the crystalline amifostine trihydrate is thermally stable atabout ambient temperature for at least two years.
 30. The method ofclaim 22 where the dosage form is lyophilized.
 31. The method of claim22, wherein the dosage form further comprises an excipient.
 32. Themethod of claim 31, wherein the excipient is mannitol.
 33. The method ofclaim 22, wherein the dosage form comprises 500 mg of amifostine on ananhydrous basis.
 34. The method of claim 22, wherein the amount of thecrystalline compound administrated is from about 10 to 1000 mg/m². 35.The method of claim 22, wherein the amount of the crystalline compoundadministrated is from about 200 to 750 mg/m².
 36. The method of claim22, wherein the amount of the crystalline compound administrated isabout 750 mg/m².
 37. The method of claim 22, wherein the dosage form isadministrated before the radiation therapy.
 38. The method of claim 22,wherein the dosage form is administrated after the radiation therapy.39. The method of claim 22, wherein the dosage form is administratedduring the radiation therapy.